A hallmark of Parkinson's disease (PD) is the accumulation of abnormally folded, highly ubiquitinated proteins in Lew body of the ventral midbrain dopamine (DA) neurons. It is well- established that accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates stereotypic stress responses and intracellular signaling pathways, collectively known as the unfolded protein response (UPR), which leads to perturbation of neuronal functions and cell death. Among the three distinct pathways activated by ER stress, IRE11 governs the most phylogenetically conserved UPR mechanism, and critically regulates the survival and cell death decision in the mammalian cells. One major mechanism for IRE11 is to activate cell death mechanism through the stress kinase pathway, which involves apoptosis signal-regulating kinase 1 (ASK1) and c-jun N-terminal kinase (JNK). However, the detailed mechanism that activates this pathway is poorly understood. Our laboratory has shown that homeodomain interacting kinase 2 (HIPK2), a member of the MAPKK family, regulates programmed cell death (PCD) during neuronal development. Interestingly, our recent results indicate that ER stress induces protein complex formation between ASK1, HIPK2 and JNK, and that the activation of HIPK2 by ASK1 is required to activate JNK. Consistent with these results, loss of HIPK2 renders neurons more resistant to ER stress-induced cell death, whereas over-expression of HIPK2 enhances neuronal death in several ER stress-related paradigms. In addition, microarray analyses in Hipk2 mutants identify mitochondrial membrane permeability transition pore, cyclophilin D, as a potential candidate target of HIPK2. These results lead to the hypothesis that ASK1-HIPK2-JNK signaling pathway connects ER stress-induced cell death signals. To test this hypothesis, we propose to (1) characterize the molecular mechanism and the specificity of IRE11-ASK1-HIPK2 signaling pathway in ER stress-induced neuronal degeneration; (2) characterize the role of cyclophilin D as the potential HIPK2 downstream target in ER stress- induced cell death; and (3) characterize the role of HIPK2 in regulating the mitochondrial dynamics under ER stress-induced cell death. These approaches are supported by strong preliminary results and use multidisciplinary approaches to reveal previously unrecognized functions of ER stress signaling in the survival of DA neurons. Our results will bring further insights to the mechanisms of ER stress in PD, and provide novel therapeutic targets for the treatment of this devastating disease. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting the general population and the veterans. An important neuropathological hallmark of PD is the accumulation of abnormally folded, highly ubiquitinated proteins (Lewy body) in the dopamine (DA) neurons, which activates endoplasmic reticulum (ER) stress responses and intracellular signaling pathways, collectively known as the unfolded protein response (UPR), leading to the perturbation of neuronal functions and cell death. In this application, we propose an array of interdisciplinary approaches to investigate the mechanism of IRE11-ASK1-HIPK2 signaling pathway that contributes to the survival and cell death in a mouse model of PD. Results from this project will provide new insights into the ER stress-dependent cell death of ventral midbrain DA neurons and new therapeutic targets to treating patients with PD.